Introduction

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Several forms of experimental and clinical hypertension are associated with oxidative stress. For instance, we have found increased reactive oxygen species (ROS) activity in rats with lead-induced hypertension (Gonick et al., 1997; Vaziri et al., 1997, 1999; Ding et al., 1998) and rats with chronic renal failure (Vaziri et al., 1998b). In addition, oxidative stress has been demonstrated in rats with cyclosporine-induced hypertension (Lopez-Ongil et al., 1998; Navarro-Antolin et al., 1998), spontaneously hypertensive rats (Tschudi et al., 1996; Schnackenberg et al., 1998; Schnackenberg and Wilcox, 1999; Vaziri et al., 2000a), Dahl salt-sensitive rats (Atarashi et al., 1997; Swei et al., 1997), diet-induced hypertension (Roberts et al., 2000, 2001), patients with essential hypertension (Kumar and Das, 1993; Lacy et al., 1998), and women with preeclampsia (Roggensack et al., 1999). Oxidative stress can potentially contribute to generation and maintenance of hypertension via inactivation of NO (Vaziri et al., 1997, 1998b, 1999; Schnackenberg et al., 1998), a nonenzymatic generation of vasoconstrictive isoprostanes from arachidonic acid peroxidation (Takahashi et al., 1992; Schnackenberg and Wilcox, 1999) and a direct vasopressor action (Atzori et al., 1991; Tesfamariam and Cohen, 1992). In fact, antioxidant administration has been shown to improve NO metabolism and ameliorate hypertension in rats with lead-induced hypertension (Vaziri et al., 1997, 1999), chronic renal failure (Vaziri et al., 1998b), and spontaneous hypertension (Schnackenberg et al., 1998; Schnackenberg and Wilcox, 1999; Vaziri et al., 2000a). However, due to the presence of numerous concurrent biochemical, hemodynamic, and/or genetic disturbances that can contribute to the development of hypertension in these models, it is difficult to attribute the associated hypertension to a direct effect of oxidative stress per se. To test the hypothesis that oxidative stress per se can cause hypertension, recently Vaziri et al. (2000b) carried out a study in which normal Sprague-Dawley rats were subjected to oxidative stress by glutathione (GSH) depletion using the GSH synthase inhibitor buthionine sulfoximine (BSO) for 2 weeks. The BSO-treated group showed a dramatic fall in tissue GSH content, marked elevation in blood pressure, and a significant reduction in urinary excretion of NO metabolites, nitrate plus nitrite (NOx), suggesting depressed NO bioavailability. This was associated with a significant accumulation in various tissues of nitrotyrosine, which is the footprint of NO inactivation by ROS. They further demonstrated a marked amelioration of hypertension together with improved urinary NOx excretion and reduced nitrotyrosine accumulation (despite GSH depletion) in the BSO-treated animals by concomitant antioxidants therapy, supporting the notion that oxidative stress was involved in the pathogenesis of hypertension in this model.

The reduction in urinary NOx excretion may be due to either diminished NO production and/or enhanced NO sequestration. With respect to the latter possibility, the increased nitrotyrosine accumulation, a secondary by-product of interactions of NO, ROS, and tyrosine residues of proteins (Eiserich et al., 1995a, 1998; Halliwell, 1997), reflects ROS-mediated NO inactivation and sequestration in this model. A second possible mechanism for depressed urinary NOx excretion in this model is diminished NO generation. This can in turn be due to diminished L-arginine availability, quantitative NO synthase (NOS), deficiency, NOS inhibition, or loss of renal parenchyma (Vaziri et al., 1998a). This study was intended to examine renal histology, NOS protein expression, and nitrotyrosine distribution in rats rendered hypertensive by oxidative stress using glutathione depletion.

	Materials and Methods

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Animal Model

Male Sprague-Dawley rats with an average weight of 275 g (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were housed in a climate-controlled, light-regulated space with 12-h day (500 lux) and night (<5 lux) cycles. They were fed a low-nitrate rat chow and water ad libitum. The rats were randomly assigned to either the oxidative stress group or the placebo-treated control group. The drinking water in the former group was supplemented with the GSH synthase inhibitor BSO (Sigma Chemical Company, St. Louis, MO), 30 mM/l (BSO-treated group) for 2 weeks (Vaziri et al., 2000b). This treatment was intended to raise ROS activity by depleting GSH, which is a major component of the natural antioxidant defense system. The control group was provided with regular water.

After 2 weeks of BSO or placebo treatments and under general anesthesia (Nembutal; 50 mg/kg i.p.), the animals were killed by exsanguination using cardiac puncture. Kidneys were immediately removed, frozen in liquid nitrogen, and stored at 70°C until processed. In addition, plasma was separated and stored at 70°C. During the observation period, tail arterial blood pressure was measured and timed urine collections were obtained using individual metabolic cages. Hematocrit and serum and urine creatinine concentrations were measured by standard techniques.

Measurement of Blood Pressure

Arterial blood pressure was measured by tail plethysmography (Harvard Apparatus, Inc., Natick, MA), as previously described (Vaziri et al., 1998a, 2000b). Briefly, conscious rats were placed in a restrainer on a heated pad and allowed to rest inside the cage for 15 min before blood pressure measurements. The procedure was carried out in a climate-controlled room with an ambient temperature of 70°F. Rat tails were placed inside a tail cuff, and the cuff was inflated and released several times to allow conditioning of the animals to the procedure. A minimum of four consecutive measurements were taken and recorded by a student oscillograph (Harvard Apparatus). The data were then averaged for presentation.

Tissue Glutathione Assay

Total GSH content of the hepatic tissue was determined using a Cayman GSH assay kit (Cayman Chemical Co., Ann Arbor, MI). The assay uses a carefully optimized enzymatic recycling method using GSH reductase whereby the sulfhydryl group of GSH reacts with 5,5'-dithio-bis-2-nitrobenzoic acid and Ellman's reagent producing a yellow colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide, GSTNB (between GSH and TNB), which is concomitantly produced, is reduced by GSH reductase to recycle GSH and produce more TNB. The rate of TNB production is directly proportional to this recycling reaction, which is in turn directly proportional to the concentration of GSH in the sample. Thus, measurement of TNB at 405 or 412 nm provides an accurate estimate of GSH present in the sample. It should be noted that oxidized GSH is converted to GSH by GSH reductase in this system, which consequently measures total GSH.

Total Nitrate and Nitrate NOx Assay

Urinary excretion of the total nitrate and nitrite was determined as described in our previous studies (Vaziri et al., 1998a; Zhou et al., 2000a,b) using the purge system of a Sievers Instruments model 270B nitric oxide analyzer (NOA; Sievers Instruments, Inc., Boulder, CO).

Histologic Examinations. Six animals in each group were exsanguinated for morphological studies. Immediately after exsanguination, the kidneys were removed, promptly sectioned, and separately postfixed in 10% buffered formalin. The fixed tissue was embedded in paraffin and sectioned. The sections were stained with Masson's trichrome stain, periodic acid-Schiff stain, and hematoxylin and eosin and were examined under light microscopy.

Immunoperoxidase Studies. Immunohistochemical staining for eNOS, iNOS, and nitrotyrosine were performed using standardized streptavidin-biotin-peroxidase method on formalin-fixed, paraffin-embedded renal tissues, as described previously (Zhou et al., 2000a,b). Except the incubation with the primary antibodies, all incubations were at room temperature and were separated by washes with phosphate-buffered saline. After deparaffinization, sections were treated with 1.25% hydrogen peroxide to block endogenous peroxidase activity. After preincubation with 10% normal horse or swine serum for 20 min, sections were incubated with primary antibodies overnight at 4°C, followed sequentially with biotinylated horse anti-mouse (Vector Laboratories, Burlingame, CA) or biotinylated swine anti-rabbit (Dako Corporation, Carpinteria, CA) antibodies for 20 min and streptavidin-peroxidase complex (DAKO, Carpinteria, CA) for 30 min. The working concentrations for the mouse anti-eNOS monoclonal antibody (Transduction Laboratories, Lexington, KY), rabbit anti-iNOS antibody (Transduction), and rabbit anti-nitrotyrosine antibody (Upstate Biotechnology, Inc., Lake Placid, NY) were 0.1, 0.05, and 5.9 µg/ml, respectively. For negative controls, a monoclonal mouse IgG1 (Bethyl Laboratories, Inc., Montgomery, TX) or normal rabbit serum was used at equivalent concentrations. Diaminobenzidine (Sigma Chemical Company) was used as chromogen, and hematoxylin was used for nuclear counterstain. To further verify the specificity of immunostaining for eNOS and iNOS, antibodies from different vendors (eNOS from Upstate Biotechnology; iNOS from Cayman Chemicals) were used, and immunostaining was repeated.

Data Analysis. Data were presented as mean ± S.E.M. Student's t test and regression analysis were used in evaluation of the data as appropriate. P values equal to or less than 0.05 were considered significant.

	Results

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General Data and Biochemical Measurements (Table 1). The BSO-treated animals exhibited a 3-fold reduction in total GSH content of the liver tissue and a marked rise in arterial blood pressure. No significant difference was found either in creatinine clearance or hematocrit between the two groups. The BSO-treated animals showed a sharp fall in urinary NOx excretion. A negative correlation was found between arterial blood pressure and urinary NOx excretion in the study groups (r = 0.77; p < 0.001).

Renal Histology (Fig. 1). The morphological appearance of glomeruli, arteries, arterioles, and tubulointerstitium in BSO-treated animals was normal and indistinguishable from that of the control animals.

View larger version (134K): [in this window] [in a new window]   Fig. 1.   Photomicrographs of representative sections of renal cortex from control (a) and BSO-treated (b) animals. The morphological appearance of glomeruli, arteries, arterioles, and tubulointerstitium was normal in both groups.

Immunoperoxidase Studies for eNOS and iNOS. The intensities and distributions of eNOS were similar between the two groups (Fig. 2, a and b). In both groups, there was mild to moderate staining of endothelial and smooth muscle cells in the small arteries and arterioles. Focally, the cortical proximal and distal tubules also showed positivity for eNOS in both groups. However, some collecting ducts and S3 segments revealed mild eNOS positivity in the BSO-treated group, which showed slightly less intensity in the control group.

View larger version (182K): [in this window] [in a new window]   Fig. 2.   Tissue expressions of eNOS, iNOS, and nitrotyrosine in the kidneys. eNOS: scattered eNOS immunoreactivity was seen in proximal and distal tubules in both control (a) and BSO-treated (b) animals with similar intensity. iNOS: photomicrographs depict iNOS immunoreactivity in the collecting ducts. The staining pattern and intensity was comparable between the control (c) and BSO-treated (d) rats. Nitrotyrosine: the control animals (e) showed weak and focal immunoreactivity for nitrotyrosine in cortical tubular epithelial cells. In contrast, strong and diffuse positivity were observed in the cortical tubules in BSO-treated group (f).

Immunoperoxidase Studies for Nitrotyrosine. In normal control rats (Fig. 2e), mild nitrotyrosine expression was seen in some of the proximal and distal tubules of renal cortex (1+). The S3 segments of the proximal tubules were diffusely positive (1+). Collecting ducts in the inner stripe of the outer medulla and in the inner medulla were also positive (1+). Mild (1+) smooth muscle staining was observed in some of the arterial and arteriolar walls. The endothelial cells of vasa recta were mildly positive (1+). In addition, there was scattered staining (1+) in some of the glomerular visceral epithelial and endothelial cells. The glomerular mesangial cells were essentially negative.

	Discussion

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GSH is an endogenous and ubiquitous sulfhydryl-containing tripeptide that is synthesized by two enzymes (acting in concert), -glutamylcysteine synthase and GSH synthase (Poot et al., 1995). GSH has been implicated in a variety of cellular processes, including prevention of free radical formation and accompanying tissue injury (Yu, 1994). BSO inhibits -glutamylcysteine synthase irreversibly and consequently depletes tissue GSH content (Griffith and Meister, 1979). In the present study, administration of BSO led to a marked reduction in tissue GSH content and a significant increase in tissue nitrotyrosine, which is a strong indicator of NO oxidation by ROS. Induction of oxidative stress by GSH depletion with BSO resulted in a marked elevation of arterial blood pressure in otherwise intact genetically normotensive animals. These observations confirmed the results of our earlier studies (Vaziri et al., 2000b). In addition, the serum creatinine concentration and creatinine clearance were identical in the BSO-treated and placebo-treated groups. Furthermore, no morphologic abnormalities were observed in the kidneys by light microscopy in the BSO-treated rats. These observations suggest that oxidative stress-induced hypertension in this model is not associated with discernible renal disease and/or structural abnormalities of the kidney.

The rise in blood pressure in BSO-treated animals was associated with a marked reduction in urinary NOx excretion. This phenomenon could be due to depressed NO production capacity. To this end, we studied renal eNOS and iNOS expressions by immunohistochemical methods. We found no significant differences in either intensities or localizations of eNOS or iNOS signals by immunohistochemical methods. In addition, the eNOS and iNOS distributions in normal rats were similar to those described earlier (Kone, 1999; Zhou et al., 2000a,b). Therefore, the reduction in urinary NOx excretion cannot be attributed to a quantitative NOS deficiency. Likewise, it was not due to reduced dietary L-arginine content because food intake and body weight were similar in the two groups.

A plausible explanation for the decline in urinary NOx excretion in the face of normal NOS isotype expressions is the potential ROS-mediated oxidation and sequestration of NO. In fact, we found a marked increase in renal tissue nitrotyrosine (the footprint of NO interaction with ROS) by immunohistochemical methods, which confirmed the results of our previous studies using Western blot analysis (Vaziri et al., 2000b). It is of note that generation of NO from L-arginine by NOS depends on the presence of the NOS cofactor tetrahydrobiopterin, which itself is readily oxidized by ROS. Thus, in addition to direct oxidation/inactivation of NO, oxidative stress can limit NO production by NOS via depletion of its cofactor tetrahydrobiopterin.

Interaction of ROS, particularly that of superoxide with NO, leads to the production of peroxynitrite, which is a highly cytotoxic reactive compound (Beckman and Koppenol, 1996; Halliwell, 1997). Peroxynitrite can in turn react with DNA, lipid, and protein molecules (Halliwell, 1997). For instance, peroxynitrite reacts with the tyrosine residues in various proteins to produce nitrotyrosine. Alternatively, ROS can initially activate tyrosine residues to produce tyrosyl radicals that can in turn oxidize NO to produce nitrotyrosine (Eiserich et al., 1995b). In addition, nitrotyrosine can be formed from the interaction of tyrosine with other reactive nitrogen species (Eiserich et al., 1995b; Halliwell, 1997). However, the contribution of the latter reactions to total tissue nitrotyrosine abundance is limited. As a result, nitrotyrosine abundance is largely a function of ROS interaction with NO (Beckman et al., 1994; Beckman and Koppenol, 1996). The observed accumulation of nitrotyrosine in the BSO-treated animals points to the effectiveness of BSO in generating the intended oxidative stress in the study animals. In addition, the increased tissue nitrotyrosine burden was indicative of the inactivation and sequestration of NO. This could contribute to the reduction of urinary NOx excretion and NO bioavailability. Reduced NO bioavailability resulting from enhanced NO inactivation by ROS could in turn contribute to the pathogenesis of hypertension in the BSO-treated animals. The role of oxidative stress in the pathogenesis of hypertension in this model is further supported by the efficacy of concomitant antioxidant therapy in our earlier study (Vaziri et al., 2000b). We demonstrated that concomitant antioxidant therapy with vitamin E plus vitamin C prevented BSO-induced renal nitrotyrosine accumulation (by Western blot analysis) and ameliorated hypertension without effecting the associated GSH deficiency. These observations point to the role of oxidative stress in the pathogenesis of hypertension as opposed to an unrelated effect of BSO.

Nitrotyrosine was primarily distributed in the proximal tubules, distal tubules, and collecting duct. These observations are consistent with a previous report by Bian et al., (1999). The above structures heavily depend on oxidative metabolism for energy-dependent sodium transport. In contrast, nitrotyrosine abundance was minimal in the glomeruli in which H₂O and solute transport is passive. Generation of superoxide by mitochondria is a function of oxygen consumption in the given tissue. Thus, high oxygen consumption by renal tubules is predictably coupled with a high superoxide generation, which in the presence of NO (since the tubule epithelium is the location for both eNOS and iNOS) and depressed natural antioxidant capacity (GSH depletion) can lead to local oxidative stress and nitrotyrosine formation. This is clearly evidenced by the intense accumulation of nitrotyrosine in those locations in the BSO-treated animals. Another source of nitrotyrosine may be the reabsorption of nitrated proteins by the endocytic lysosomal apparatus of the proximal tubules. In fact, we found that the strongest staining of nitrotyrosine is not in the basal mitochondria region but rather in the area underneath the luminal brush borders where the endocytic lysosomal apparatus is distributed. Thus, the cellular location of nitrotyrosine staining suggests that a large portion of nitrotyrosine may come from the reabsorption of nitrated proteins (Bian et al., 1999).

In conclusion, we have confirmed the results of our earlier study (Vaziri et al., 2000b) in which we stated that chronic oxidative stress per se can lead to enhanced NO inactivation and induction of severe hypertension in genetically normotensive rats. We have further shown that eNOS and iNOS expressions and renal morphology are normal in this model, suggesting that oxidative stress-induced hypertension is not caused by discernible renal injury or quantitative NOS deficiency in this model. Instead, depressed urinary NOx excretion and increased nitrotyrosine accumulation in the face of normal NOS isoform expression point to enhanced ROS-mediated NO inactivation and functional NO deficiency in BSO-treated animals.